Cell-proliferation inhibiting vpg proteins, fragments or analogs thereof and their applications

ABSTRACT

Use of VPg proteins, fragments or analogs thereof having the ability to bind an eukaryotic initiation factor eIF4E, for inhibiting cell-proliferation.

The present invention relates to the field of cellproliferation-inhibiting proteins. More particularly, the inventionrelates to a protein comprising or consisting of a VPg protein,fragments thereof or analogs thereof, that are cytotoxic to eukaryoticcells, and/or inhibit the cell proliferation. The invention also relatesto medical uses of such a protein, fragments thereof or analogs thereof.

Eukaryotic initiation factor eIF4E is a mRNA 5′ cap-binding protein,which both attaches mRNA and binds the eIF4G component of the eIF4Finitiation complex. A significant fraction of eIF4E (up to nearly 70%)localizes to the nucleus (Lejbkowicz et al., 1992). It has been shownthat nuclear eIF4E is involved in the nucleocytoplasmic transport ofcertain messenger RNAs (Lejbkowicz et al., 1992; Rosenwald et al., 1995;Rousseau et al., 1996; Lai and Borden, 2000). It has been demonstratedthat the unchecked over-expression of eIF4E can cause cell growth andmalignant transformation, whereas reduction of the eIF4E level canreverse the transformed phenotype (reviewed in Sonenberg and Gingras,1998). Furthermore, in humans, some tumor types exhibit elevated levelsof eIF4E (Ruggero and Pandolfi, 2003). It appears that at high eIF4Elevels there is increased cytoplasmic level of proteins involved in themalignant transformation, due to the increase in the eIF4E-dependenttransport of the appropriate mRNAs to the cytoplasm (Shantz and Pegg,1994; Rousseau et al., 1996; Lai and Borden, 2000).

International Application No. WO 00/78803 describes eIF4E-bindingpeptides useful in therapy, and particularly for the induction ofprogrammed cell death, presumably by disrupting the formation of theeIF4F complex. All these peptides comprise the amino acid sequence(motif) YxxxxLØ (wherein x is any amino acid and Ø is Leu, Met or Phe),required for binding human eIF4E (accession number NP_(—)001959 in theGENBANK database, Apr. 15, 2007 version).

Thus, there is an interest in the development of adequate eIF4E bindingagents for use in anti-cancer therapy. There is also a need to findeIF4E binding agents possessing a distinct mechanism of action from thatdescribed above, to overcome drug resistances for instance.

Potato virus Y (PVY) is the type member of the Potyvirus genus,belonging to the Potyviridae family, the largest and economically mostimportant group of plant viruses. Potyviruses have a so-calledgenome-linked VPg protein (hereinafter referred to as VPg protein orVPg) covalently attached to the 5′ end of their linear nucleic acidgenomes (Riechmann et al., 1989 and Murphy et al., 1991). The potyvirusVPg protein is implicated in virus protein synthesis, long-distancemovement in plant tissue and virus replication (Lellis et al., 2002;Schaad et al., 2002; Fellers et al., 1998). Early after potyvirusinfection, the genomic RNA interacts with the host cellular machinery toinitiate protein synthesis. The VPg protein seems to play an importantrole in this early event, because removal of VPg protein with proteinaseK from the genomic RNA abolishes RNA infectivity and, in addition,decreases its translation efficiency in vitro (Leonard et al., 2000;Herbert et al., 1997). The amino acid sequence of the potato virus Ygenome-linked protein VPg corresponds to the amino acid residues 336 to523 of the amino acid sequence identified under accession numberCAA82642 in the GENBANK database (Nov. 14, 2006 version). It isreproduced herein as SEQ ID NO: 2.

Recent studies have reported an interaction between the VPg protein ofcertain potyviruses (turnip mosaic virus, lettuce mosaic virus, tobaccovein mottling virus, potato virus Y) and the plant translationinitiation factor eIF4E (Wittmann et al., 1997; Duprat et al., 2002;Kang et al., 2005; Grzela et al., 2006). It has also been shown that theVPg protein encoded by certain vertebrate viruses of the Caliciviridaefamily, such as the feline calcivirus (FCV), the lordsdale virus (LDV))or the recently discovered murine norovirus (MNV-1) interact with thecap-binding protein eIF4E (Goodfellow et al., 2005; Chaudhry et al.,2006).

Grzela et al. (2006) have found that the interaction between PVY VPgprotein and eIF4E seems to have rather low species specificity since itwas also observed with yeast as well as with the insect (S. frigiperda)eIF4Es. The authors have also described that the VPg/eIF4E interactionresults in some inhibition of cell-free protein synthesis in insectcells, but without leading to insect cell death. However, the PVY VPgprimary amino acid sequence does not exhibit the eIF4E-binding motifYxxxxLØ described above.

Now, the Inventors have observed that, in presence of potato virus Y VPgprotein, the nuclear pool of eIF4E was dramatically depleted in humancells. Thus and surprisingly, the Inventors have shown that the VPgprotein immobilizes human initiation factor eIF4E in the cytoplasm,which results, contrary to what was observed previously in insect cells,in the inhibition of cell proliferation, leading to cell death.

Therefore, in a first aspect, the present invention relates to anisolated and purified protein comprising or consisting of a VPg proteinor a fragment thereof of at least 8 amino acids or an analog thereof:

(i) having the ability to bind an eukaryote initiation factor eIF4E,preferably a mammal initiation factor eIF4E and more preferably a humaninitiation factor eIF4E, and

(ii) said protein, fragment thereof or analog thereof not comprising theamino acid motif YxxxxLØ, wherein x is any amino acid, Y is tyrosine(Tyr), L is leucine (Leu) and Ø is leucine (Leu), methionine (Met) orphenylalanine (Phe), as a medicament, preferably as an anti-canceragent, or for use in treating cancer, preferably glioma, melanoma or acolon or lung cancer, in which the cancer cells express, preferablyover-express, the initiation factor eIF4E, in a subject.

A “subject” refers to a mammal, preferably a human.

The characterization of the binding (or interaction) of a protein, afragment thereof or an analog thereof according to the present inventionto an eukaryotic (e.g., insect), preferably a mammal and more preferablya human eIF4E can be performed by an ELISA-based binding assay asdescribed in Grzela et al. (2006). Advantageously, the dissociationconstant between said protein, fragment thereof or analog thereof andsaid eIF4E is between about 5 nM and about 450 nM. Dissociation constant(or affinity) measurements may be made using methods known to thoseskilled in the art, including using the method described in Grzela etal. (2006). Optionally, this characterization may be combined with thein vitro determination of the change in cellular localization of eIF4E(immobilization of eIF4E in the cytoplasm) in the presence of a protein,a fragment thereof or an analog thereof of the present invention, ineukaryotic, preferably mammal and more preferably human cells. By way ofexample, the in vitro determination of the change in cellularlocalization of eIF4E in the presence of a protein, a fragment thereofor an analog thereof of the present invention can be performed in insectcells (e.g., High Five (Trichoplusia ni) cells), since these cells are agood predictive model for said determination in human cells. Thisdetermination can be performed as in Examples 3 or 4 below.

The terms “protein” and “peptide” may be used interchangeably herein.They are well known in the art and refer to a polymeric form of aminoacids of any length. The terms also refer to a protein or peptidecomprising chemically or biochemically modified amino acids, as well asa protein or peptide having modified peptide backbones, e.g. a proteinor peptide containing one or more modifications to L-amino acidside-chains (for example D-amino acids), or to the alpha-amino acidbackbone.

A VPg protein, according to the present invention, refers usually to avirus-encoded protein covalently attached to the 5′ end of the linearnucleic genome of said virus (Riechmann et al., 1989 and Murphy et al.,1991), and also comprises according to the present invention:

-   -   an isolated plant virus-encoded VPg protein,    -   a recombinant VPg protein of a plant virus, or    -   a synthetic VPg protein of a plant virus.

Preferably, said VPg protein consists of about 188 to 193 amino acidresidues and includes the amino acid motif KGK and NMYG (SEQ ID NO: 26).

In a preferred embodiment, the VPg protein is the potato virus Y-encodedVPg protein of SEQ ID NO: 2.

The term “plant virus” refers to a virus capable of spreading through aplant and particularly of infecting at least one type of plant cells.

In another embodiment of the present invention, the VPg protein analogis an analog of PVY VPg protein of SEQ ID NO: 2, exhibiting at least20%, and by order of increasing preference, at least 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%sequence identity, or at least 45%, and by order of increasingpreference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98% or 99% sequence similarity with the full-length of PVY VPg proteinof SEQ ID NO: 2, provided that said VPg protein analog still has theability to bind an eukaryote (e.g., insect) initiation factor eIF4E,preferably a mammal initiation factor eIF4E and more preferably a humaninitiation factor eIF4E as described above.

Examples of VPg protein analogs are VPg proteins encoded:

-   -   by other plant virus than the PVY, preferably belonging to the        Potyviridae family, more preferably to the Potyvirus genus, such        as the tobacco etch virus (TEV) (SEQ ID NO: 4), the clover        yellow vein virus (C1YVV) (accession number NP_(—)734169.1 in        the GENBANK database, Mar. 30, 2006, version, reproduced herein        as SEQ ID NO: 6), the tobacco vein mottling virus (TVMV)        (accession number NP_(—)734333.1 in the GENBANK database, Mar.        30, 2006, version, reproduced herein as SEQ ID NO: 8), the        turnip mosaic virus (TuMV) (SEQ ID NO: 10), the lettuce mosaic        virus (LMV) (accession number NP_(—)734159.1 in the GENBANK        database, Mar. 30, 2006, version, reproduced herein as SEQ ID        NO: 12), or    -   by a virus belonging to the Calcivirus family, preferably to the        Norovirus genus, such as the feline calicivirus (FCV) (accession        number Q66914 in the UniProtKB/Swiss-Prot database, reproduced        herein as SEQ ID NO: 27), human norovirus (HuNoV, accession        numbers P54634 in the UniProtKB/Swiss-Prot database, and        NP_(—)056820 in the GENBANK database, reproduced herein as SEQ        ID NO: 28 and 29 respectively) and murine norovirus (MNV)        (accession number YP_(—)720001 in the GENBANK database,        reproduced herein as SEQ ID NO: 30).

According to another embodiment of the present invention, the fragmentof a protein as defined above is a fragment of a VPg protein or afragment of a protein analog of a VPg protein as defined here above,comprising or consisting of at least 8, and by order of increasingpreference at least 10, 15, 20, 25, 26 or 42 contiguous amino acidsresidues of said VPg protein or said analog thereof, but that retainsthe ability to bind an eukaryote (e.g., insect) initiation factor eIF4E,preferably a mammal initiation factor eIF4E and more preferably a humaninitiation factor eIF4E, as described above.

By way of example, the fragment as defined above comprises or consistsof at least 8, and by order of increasing preference at least 10, 15,20, 25, 26 or 42 contiguous amino acids residues of the amino acidsequence of any of the VPg protein as defined here above, preferably ofSEQ ID NO: 2, 4, 6, 8, 10 or 12.

In another preferred embodiment of the present invention, the fragmentof PVY VPg (SEQ ID NO: 2) comprises or consists of the amino acidsequence located:

-   -   between the arginine (Arg) at position 41 and the arginine (Arg)        at position 94 (also named VPg2; SEQ ID NO: 14) of SEQ ID NO: 2,        or    -   between the arginine (Arg) at position 41 and the glycine (Gly)        at position 82 (also named VPg4; SEQ ID NO: 16) of SEQ ID NO: 2,    -   between the arginine (Arg) at position 41 and the phenylalanine        (Phe) at position 66 (also named VPg5; SEQ ID NO: 18) of SEQ ID        NO: 2,    -   between the arginine (Arg) at position 41 and the arginine (Arg)        at position 59 (also named VPg1; SEQ ID NO: 20) of SEQ ID NO: 2,        or    -   between the phenylalanine (Phe) at position 60 and the arginine        (Arg) at position 94 (also named VPg3; SEQ ID NO: 22) of SEQ ID        NO: 2.

In another embodiment, the fragment comprises or consists of at least 8,and by order of increasing preference at least 10, 15, 20, 25 or 26contiguous amino acids residues of the amino acid sequence SEQ ID NO:16.

In another embodiment, the fragment of the VPg protein consists of ananalog of SEQ ID NO: 16 exhibiting at least 60%, and by order ofincreasing preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99% sequence identity, or at least 80%, and by order of increasingpreference, at least 85%, 90%, 95%, 98% or 99% sequence similarity withthe full-length of amino acid sequence of SEQ ID NO: 16.

In another embodiment, the fragment of the VPg protein consists of ananalog of SEQ ID NO: 18 exhibiting at least 60%, and by order ofincreasing preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99% sequence identity, or at least 80%, and by order of increasingpreference, at least 85%, 90%, 95%, 98% or 99% sequence similarity withthe full-length of amino acid sequence of SEQ ID NO: 18.

The term “analog” is intended to encompass any form of PVY VPg protein(SEQ ID NO: 2) or a fragment thereof (e.g., SEQ ID NO: 16 or SEQ ID NO:18) as defined here above, wherein one or more of the amino acids withinthe amino acid sequence has been replaced with an alternative amino acidor another residue such as a carbohydrate, a lipid or a chemical and/orwherein one or more of the amino acids has been deleted or wherein oneor more additional amino acids or another residue such as acarbohydrate, a lipid or a chemical has been added to the peptide chainor amino acid sequences of PVY VPg protein or a fragment thereof,provided that said analog still has the ability to bind an eukaryote(e.g., insect) initiation factor eIF4E, preferably a mammal initiationfactor eIF4E and more preferably a human initiation factor eIF4E, asdescribed above.

Many suitable computer programs for calculating the “identity” betweentwo amino acid sequences are generally known in the art, such as BLASTprogram (http://www.ncbi.nlm.nih.gov/BLAST/) choosing the scoring matrixBLOSUM62.

The term “similarity” refers to a comparison of the amino acid residuesof two peptides wherein conservative amino acid residue substitutionsare permitted, i.e. amino acid residues that share the same charge andthe same polarity are considered similar. The conservative changes donot significantly alter the binding characteristics of the resultingpeptide. The following is one example of various groupings of aminoacids:

Amino Acids with Nonpolar R Group

Alanine, Valine, Leucine, Isoleucine, Proline, Plenylalanine,Tryptophan, Methionine.

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine.

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)Asapartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0) Lysine, Arginine,Histidine (at pH 6.0)

Amino Acids with OH Group

Serine, Threonine, Tyrosine

Amino Acids with an Aromatic Group

Phenylalanine, Tyrosine, Tryptophan

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asaparagine 132 Aspartic acid133 Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine(at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan204

Particularly preferred conservative substitutions are:

-   -   Lysine for Arginine and vice versa such that a positive charge        may be maintained;    -   Glutamic acid for Aspartic acid and vice versa such that a        negative charge may be maintained;    -   Serine for Threonine such that a free —OH can be maintained;    -   Tyrosine for Phenylalanine such that the aromatic character of        the residue can be maintained; and    -   Glutamine for Asparagine such that a free —NH₂ can be        maintained.

The sequence similarity values provided herein are determined asdescribed above in reference to calculation of sequence identity.

Optionally, the protein, fragment thereof or analog thereof as definedhere above may comprise an amino acid sequence facilitating cellularuptake. Such amino acid sequences, known as Cell Penetrating Peptides,are well known in the art; See CELL PENETRATING PEPTIDES: PROCESSES ANDAPPLICATIONS, edited by Ulo Langel (2002); or Advanced Drug DeliveryReviews, 2005, 57:489-660). These include the Human ImmunodeficencyVirus type 1 (HIV-1) protein Tat or fragment thereof (Ruben et al.,1989), the herpes virus tegument protein VP22 (Elliott and O'Hare,1997), penetratin (Derossi et al., 1996), protegrin 1 anti-microbialpeptide SynB (Kokryakov et al., 1993) and the basic fibroblast growthfactor (Jans, 1994).

A second aspect of the present invention relates to the use of aprotein, a fragment thereof or an analog thereof as defined here abovefor the manufacture of a medicament for treating cancer, preferablyglioma, melanoma or a lung or colon cancer, in which the initiationfactor eIF4E is expressed or over-expressed, in a subject.

In a third aspect, the present invention provides an isolated nucleicacid sequence encoding a protein, a fragment thereof or an analogthereof as defined above. The nucleic acid sequence of the invention canbe synthesized using standard techniques. Techniques for nucleic acidmanipulation are known in the art (See, for example, in Sambrook J. etal. (2000) Molecular Cloning: A Laboratory Manual). By way of example,the nucleic acid sequence comprises or consists of the nucleic acidsequences SEQ ID NO: 1 (residues 1007 to 1570 of the nucleotide sequenceidentified under accession number Z29526 in the GENBANK database (Nov.14, 2006 version) encoding PVY VPg protein), or SEQ ID NO: 3, 5, 7, 9,11, 13, 15, 17, 19 or 21.

The present invention also provides a recombinant vector for theexpression of a protein, a fragment thereof or an analog thereof asdefined above. The recombinant vector comprises at least one nucleicacid sequence encoding a protein, a fragment thereof or an analogthereof as defined above, operably linked to at least one regulatorysequence.

The term “vector” is intended to mean a nucleic acid molecule capable oftransporting another nucleic acid. By way of example, a vector which canbe used in the present invention includes, but is not limited to, aviral vector (e.g., retrovirus, adenovirus, baculovirus), a plasmid, aRNA vector or a linear or circular DNA or RNA molecule which may consistof a chromosomal, non chromosomal, semi-synthetic or synthetic nucleicacid. Large numbers of suitable vectors are known to those of skill inthe art and commercially available. Preferred vectors are those capableof autonomous replication (episomal vector) and/or expression of nucleicacids to which they are operably linked (expression vectors).

The term “operably linked” is intended to mean that the nucleotidesequence is linked to a regulatory sequence in a manner which allowsexpression of the nucleotide sequence.

The term “regulatory sequence” includes promoters, enhancers andtranscriptional or translational control elements. Such regulatorysequences are also known in the art.

By way of example, expression vectors may include an origin ofreplication or autonomously replicating sequence (ARS) and expressioncontrol sequences, a promoter, an enhancer and necessary processinginformation sites, such as ribosome-binding sites, RNA splice sites,polyadenylation sites, transcriptional and translational terminatorsequences, and mRNA stabilizing sequences. Such vectors may be preparedby means of standard recombinant techniques well known in the art anddiscussed, for example, in Sambrook et al., 2000.

The present invention also relates to the use of at least onerecombinant vector expressing a nucleic acid encoding a protein, afragment thereof or an analog thereof as defined above, for thepreparation of a medicament for preventing or treating cancer.

The expression vector according to the present invention can also beused to transfect or transduct (e.g., by electroporation) or infectcells to thereby introduce or produce a protein, a fragment thereof oran analog thereof as defined above.

The present invention further relates to a host cell transfected toexpress a protein, a fragment thereof or an analog thereof as definedabove. The host cell can be transfected with the nucleic acid or thevector of the present invention. The host cell may be any procaryotic oreucaryotic cell. For example, a protein, a fragment thereof or an analogthereof as defined above may be expressed in bacterial cells such as E.coli, insect cells, yeast, mammalian cells such as Chinese hamster ovarycells (CHO) or animal or human cells such as B16, LGL26, HeLa or 293 orany transformed cell line.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a protein, a fragment thereof or an analogthereof or a recombinant vector as defined here above and apharmaceutically acceptable carrier. The composition according to thepresent invention is useful for treating cancer, preferably glioma,melanoma or a colon or lung cancer, in which the initiation factor eIF4Eis expressed or over-expressed, in a subject.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, liposomes, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield. Preferred examples of such carriers or diluents include, but arenot limited to, water, saline, Ringer's solutions, dextrose solution,and 5% human serum albumin. Cationic lipids, non-aqueous vehicles suchas fixed oils and commercially available transductants may also be used.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with a protein, a fragment thereof or an analogthereof as defined hereabove, use thereof in the composition of thepresent invention is contemplated.

By way of example, when the pharmaceutical composition comprises aprotein, a fragment thereof or an analog thereof, then thepharmaceutically acceptable carrier is preferably a cationic lipid, or amixture of non-cationic lipids and a cationic lipid.

In yet another aspect, the present invention comprises a method ofinhibiting cancer cell proliferation, characterized in that said methodcomprises the step of treating a subject in need thereof with acomposition comprising a protein, a fragment thereof or an analogthereof as defined here above.

The term “inhibiting” refers to slowing, decreasing, delaying,preventing or abolishing cell proliferation.

The term “cell proliferation” refers to the rate at which a group ofcells divides. The number of cells growing can be easily quantified byone skilled in the art.

The term “cancer” refers to any of a number of diseases that arecharacterized by uncontrolled, abnormal proliferation of cells, theability of affected cells to spread locally or through the bloodstreamand lymphatic system to other parts of the body (i.e., metastases), aswell as any of a number of characteristic structural and/or molecularfeatures. Examples of cancers are breast, colorectal, liver, lung (suchas small cells, non-small cells), bronchic, prostate, ovarian, brain,pancreatic, colon, head and neck, stomach and bladder cancers,non-Hodgkin's lymphomas, melanomas, leukaemias, neuroblastomas, gliomas,or glioblastomas.

A “cancer cell” is understood as a cell having specific structuralproperties, which can lack differentiation and be capable of invasionand metastasis. Preferably, the cancer cells express or over-express theinitiation factor eIF4E. Cancer cells expressing eIF4E can be identifiedby methods known in the art, e.g., by DNA sequencing, or by Northernblot, or by immunochemistry using anti-eIF4E antibodies.

The term “over-express” means that the gene encoding eIF4E protein isexpressed at a higher level compared to the one of a normal (i.e., noncancer cell) quiescent eukaryotic, mammal or human cell, resulting inthe production in a cancer cell with eIF4E level exceeding the onenormally produced in said normal cell. The normal range of expression orproduction can be determined by routine methods as by assaying the eIF4Eprotein, its mRNA, or its gene.

The term “treating” includes the administration of the protein, afragment thereof or an analog thereof as defined here above, or acomposition of the present invention, to a patient who has a disease ordisorder (e.g., cancer or metastatic cancer), a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease, or extend duration oflife.

In another aspect, the present invention relates to the use of anisolated and purified initiation factor eIF4E, preferably from humanorigin, to screen for compounds (e.g., drugs) that mimic the bindingspecificity of a VPg protein, preferably the VPg protein of SEQ ID NO:2, with eIF4E.

In another aspect the present invention relates to a method of screeningfor compounds (e.g., drugs) which mimic the binding specificity of a VPgprotein, preferably of SEQ ID NO: 2, 4, 6, 8, 10 or 12, and morepreferably of SEQ ID NO: 2, with the initiation factor eIF4E (preferablythe human eIF4E), comprising the steps of i) contacting an isolated andpurified eIF4E with a candidate compound and a protein, a fragmentthereof or an analog thereof according to the present invention (e.g.,SEQ ID NO: 2) and ii) detecting if this candidate compound inhibits thebinding (or interaction) between eIF4E and said protein, a fragmentthereof or an analog thereof. Standard methods are available in the artto carry out competitive binding assays. In addition, the detection ofthe binding (or interaction) of a peptide compound with eIF4E can becarried out as described in Grzela et al. (2006).

Candidate compounds include peptides such as soluble peptides, lipids,carbohydrates, lipopeptides, glycopeptides, and small organic andinorganic molecules.

The Table 1 below sums up the nucleic acid and peptide sequences used inthe present description.

SEQ ID NO: Description of the sequence 1 nucleic acid sequence encodingPVY VPg protein 2 PVY VPg protein 3 nucleic acid sequence encoding TEVVPg protein 4 TEV VPg protein 5 nucleic acid sequence encoding ClYVV VPgprotein 6 ClYVV VPg protein 7 nucleic acid sequence encoding TVMV VPgprotein 8 TVMV VPg protein 9 nucleic acid sequence encoding TuMV VPgprotein 10 TuMV VPg protein 11 nucleic acid sequence encoding LMV VPgprotein 12 LMV VPg protein 13 nucleic acid sequence encoding VPg2 14VPg2: amino acid sequence located between the arginine (Arg) at position41 and the arginine (Arg) at position 94 of SEQ ID NO: 2 15 nucleic acidsequence encoding VPg4 16 VPg4: amino acid sequence located between thearginine (Arg) at position 41 and the glycine (Gly) at position 82 ofSEQ ID NO: 2 17 nucleic acid sequence encoding VPg5 18 VPg5: amino acidsequence located between the arginine (Arg) at position 41 and thephenylalanine (Phe) at position 66 of SEQ ID NO: 2 19 nucleic acidsequence encoding VPg1 20 VPg1: amino acid sequence located between thearginine (Arg) at position 41 and the arginine (Arg) at position 59 ofSEQ ID NO: 2 21 nucleic acid sequence encoding VPg3 22 VPg3: amino acidsequence located between the phenyl- alanine (Phe) at position 60 andthe arginine (Arg) at position 94 of SEQ ID NO: 2 23 PVY VPg geneforward primer 24 PVY VPg gene reverse primer 25 polyhedrin promotor ofAcMNPV 26 VPg motif NMYG 27 FCV VPg protein 28 HuNoV VPg protein 29HuNoV VPg protein 30 MNV VPg protein

In addition to the preceding features, the invention further comprisesother features which will emerge from the following description, whichrefers to examples illustrating the present invention, as well as to theappended figures.

FIG. 1: Interaction assay between human eIF4E and PVY VPg protein orfragments thereof (VPg1 to VPg4).

FIG. 2: Effect of VPg protein (SEQ ID NO: 2) expression on localizationof native elF4E in insect cells. VPg was expressed in insect cells anddistribution of native elF4E (left column) and recombinant VPg (VPcolumn) was analyzed by confocal microscopy at the indicated timepointspost infection. The blow-ups of some cells are shown in the rightcolumn.

FIG. 3: Cytoplasmic retention of native elF4E is specific to VPg protein(SEQ ID NO: 2). Insect cells have been infected with bacmid (nullbaculovirus, left column), domain II of HCV helicase (middle column) andVPg (right column). Insect cells were analyzed by confocal microscopy atthe indicated timepoints post infection.

FIG. 4: elF4E expression in fractionated insect cells. Insect cellsinfected at MOI 10 with appropriate baculoviruses were collected atindicated timepoints post infection, nuclei were separated from thecytoplasm and the subcellular extracts were analysed by SDS/PAGE andWestern blot. The expression of the recombinant proteins is shown on thefar right lower panel.

FIG. 5: Effect of PVY VPg protein on human eIF4E localisation. HeLacells were transduced with VPg in the presence of Pro-Ject™ (purchasedfrom Pierce) and analysed for presence of VPg and eIF4E by confocalmiscroscopy (FIG. 1B). In FIG. 1A the HeLa cells were not transduced.

FIG. 6: Effect of PVY VPg protein on human cell proliferation. Cellswere transduced with VPg using as protein transfer reagent TransPass P(Ozyme), and incubated for indicated periods. Cells damage was estimatedusing LDH Cytotoxicity detection assay (Clontech). The results show theaverage result obtained from three wells.

FIG. 7: Antitumour efficacy of PVY VPg protein in mice carrying GL26(mouse glioma) tumours. 0=tumour volume measured at the beginning of theexperiments. no treatment, 1=tumour volume measured 3 days later.Control=untreated mice. Buffer=mice electroporated with 0.9% NaCl.VPg=mice electroporated with 25 μg VPg in 0.9% NaCl. The same sign atthe day 0 and 3 days later shows the volume of the tumour in the samemouse. The figure also give the fold increase in tumour size betweentime 0 and time 1 (fold 1 means no increase in tumour size).

FIG. 8: Antitumour efficacy of PVY VPg protein in mice carrying B16-ova(murine melanoma) tumours. B16-ova melanoma cells were injected into thethigh of the C57BL/6 mice (two groups of 8 animals). Two weeks laterpalpable tumors were formed. Control group was injected into the tumorwith buffer mixed with Transpass P. Second group had injections of 50micrograms of VPg transduced with Transpass P. every day for 6 days. Thediameter of the tumours were measured every day during six days oftreatment.

EXAMPLES Example 1 Materials and Methods

Cells.

High-Five insect (Trichoplusia ni) cells grown in suspension werecultured in Express Five SFM medium (Invitrogen) with gentamicin (50mg/ml) and amphotericin B (0.25 mg/ml).

HeLa (human cervix carcinoma), B16-ova (mouse melanoma), GL26 (mouseglioma), IMR90 (human primary diploid fibroblasts) were grown inDMEM+GlutaMAX™-I (Gibco) medium complemented with 10% FCS.

Antibodies. Anti-his mouse monoclonal antibody (MMS-156P) was purchasedfrom BabCO (Berkeley, Calif., USA). Anti-eIF4E mouse monoclonal (P-2;sc-9976) was from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Itrecognizes mouse, rat, human and porcine eIF4E. Anti-eIF4E rabbitpolyclonal antibody against human eIF4E (purchased from JacksonImmunoResearch Laboratories) was kind gift of Nahum Sonenberg (McGillUniversity, Montreal Canada).

Anti-VPg rabbit polyclonal antibody was prepared in Elevage Scientifiquedes Dombes (Chatillon, France), using as antigen PVY hisVPg expressed inbaculovirus (described below) and was affinity purified to VPg asfollows. First an acetone powder obtained from 1 g of non-infectedinsect cells was used to remove cross-reacting antibodies from serum.Cell powder was added to the anti-VPg rabbit serum up to finalconcentration of 1%, and stored on ice overnight with gentle rolling.After centrifugation at 10 000 g for 10 min at 4° C., supernatantfraction was applied to CNBr-activated Sepharose 4B (purchased fromAmersham Bioscience) containing VPg protein immobilized according to themanufacturers instructions. Bound antibody was eluted with 100 mMglycine pH 2.4 directly into tubes containing 30 μl of 3 M Tris, pH 8.8and 20 μl 5 M NaCl.

Recombinant protein expression and purification. VPg gene of potatovirus Y (strain O, accession number Z29526 in GENBANK database) wassynthesized by PCR using plasmid pPVY15 (available under accessionnumber Z29526 in GENBANK database, Nov. 14, 2006 version) as template,with the forward primer 5′ GGGGGGGATCCATGGGGAAAATAAA-3′ (SEQ ID NO: 23)and the reverse primer 5′ CCCCCAGATCTCTATTATTCATGCTCC-3′ (SEQ ID NO: 24,permitting the expression of the protein with the accession numberCAA82642 in the GENBANK database. VPg cDNA was inserted into pFastBacHTb (purchased from Invitrogen, Carlsbad, Calif., USA) under the controlof polyhedrin promotor (SEQ ID NO: 25) of AcMNPV (O'Reilly et al., 1992)and the recombinant baculovirus expressing his-tagged VPg wasconstructed using bacmid technology (purchased from Gibco BRL). The HighFive (Trichoplusia ni) cells in suspension were infected with therecombinant baculovirus at MOI of 5 pfu/cells. Portion (100 ml) ofrecombinant baculovirus-infected HF cells was collected at 72 h p.i.suspended in 10 ml of 50 mM phosphate buffer, pH 7.0, containing 300 mMNaCl, 6 mM β-mercaptoethanol, 5% glycerol, 0.5% Tween 20 and proteaseinhibitor cocktail (Complete, purchased from Roche) and lysed by fivecycles of freezing in liquid nitrogen and thawing at 37° C. Afterclearing the extract was passed by gravity through 1 ml of Ni-NTAagarose resin (purchased from Qiagen), equilibrated with 5 ml of lysisbuffer. Bound proteins were eluted 5×500 μl of 250 mM imidazole in the50 mM phosphate buffer, pH 7.0, containing 300 mM NaCl. Fusion tag wasremoved from VPg with TEV protease for 48 h incubation at 10° C. Theresulting products were purified on 5 ml Hi-Trap Heparin column(purchased from Amersham Pharmacia). Proteins were applied on the columnin 100 mM NaCl in 50 mM phosphate, pH 7.0, washed with the same bufferuntil the OD₂₈₀ reached stable level and then eluted in the gradient ofNaCl in 50 mM phosphate, pH 7.0 (from 100 mM to 1 M). The fractionscontaining full-length hisVPg were collected, diluted and fractionatedon 5 ml 15 S-Source column (purchased from Amersham Pharmacia) underconditions used for Hi-Trap Heparin column. His-tagged domain II of HCVhelicase was expressed in HF cells after infection with the appropriatebaculovirus at MOI 5 and purified as described (Boguszewska-Chachulskaet al., 2004). All steps were analyzed on 12% SDS-PAGE by staining withCBB, using Precision Plus Protein Dual Colour (purchased from Bio-Rad)and BenchMark Prestained Protein Ladder (purchased from Invitrogen) asmolecular weight standards.

Cell fractionation. High Five (T. ni) cells at the concentration 2×10⁶cells/ml were infected with the baculoviruses expressing VPg protein orhisVPg, or domain II of HCV helicase or empty bacmid, at MOI 5. Theinfected cells were collected at the indicated times p.i. bycentrifugations at 2000 rpm for 10 min. The pellets were washed twice inPBS and resuspended in four times the packed cell volume of hypotonicbuffer (10 mM Tris, pH 7.9, 10 mM KCl, 3 mM DTT, 0.1 mM EDTA, 0.1 mMEGTA, 0.75 mM spermidine, 15 mM spermine). The cells were allowed toswell on ice for 30 min and checked with phase microscope for completebreakage. Then a 1/10 volume of restoration buffer (50 mM Tris, pH 7.9,0.75 mM spermidine, 0.15 mM spermine, 10 mM KCl, 0.2 mM EDTA, 3 mM DTT,67.7% sucrose) was added and the homogenate was layered over a 1 ml ofsucrose cushion (30% sucrose in hypotonic buffer) and centrifuged incold for 20 min at 3000 rpm. The pelleted nuclei were resuspended infour times the packed cell volume of nuclear extraction buffer (50 mMTris, pH 7.5, 0.42 mM KCl, 6 mM DTT, 0.1 mM EDTA, 10% sucrose, 5 mMMgCl₂ 20% glycerol, 0.5 mM PMSF, 3 mg leupeptin per ml). The nuclei werethen lysed by gentle rocking at 4° C. for 30 min at 4° C. Cytoplasmicand nuclear fractions were run on 15% SDS-PAGE followed by Western blotwith anti-eIF4E antibody using ECL system with X-ray film. Densitometryof eIF4E bands was performed using GelDoc 2000 (purchased from BioRad)with Quantity One software.

Cell transduction with VPg protein. Transpass P (Ozyme) was combinedwith the purified VPg (0.1-2 μg/well) in 20 mM sodium phosphate, pH 7.0,containing 150 mM NaCl or in PBS according to the manufacturerinstructions. Serum-free medium was added to the VPg/Transpass P mixtureup to 250 μl final volume. HeLa cells (10⁵/well of 24-well dish)prewashed with serum-free medium were treated with such portion ofdelivery mixture. After indicated periods of incubation at 37° C. cellswere used for studies on protein localization (confocal microscopy) andon proliferation (LDH Kit from Clontech).

Localization of elE4E and VPg in insect cells by confocal microscopy.High Five (T. ni) cells in suspension (2×10⁵ cells/ml) were infectedwith 10 MOI of baculoviruses expressing either hisVPg, or his-taggeddomain II of HCV helicase or empty bacmid. Cells were collected at 48 hp.i by centrifugation at 3000 rpm, 5 min, 4° C., followed by two washeswith 500 μl of PBS. Cells were fixed in cold 2% PFA, left for 10 min atRT, washed two times with 500 μl of PBS and resuspended in 300 μl ofPBS. Portions of 100 μl of fixed cells were applied onto clean roundcoverslips and allowed to attach while drying in a lamina flow hood.Next day cover slips were put into wells of 24-wells plate andrehydrated at room temperature with 500 μl portions of PBS. After PBSremoval, cells were permeabilized for 10 min with cold 0.1% Triton X-100in PBS, rinsed twice with PBS and blocked with 5% serum in PBS for 30min at RT. Alter 2 washes with PBS cells were incubated for 1 h at RTwith primary antibodies (anti-his or anti-human elF4E) diluted 1/100 inPBS, followed by washing and 1 h incubation with secondary antibody(anti-rabbit FITC or anti-mouse Texas Red) (Jackson ImmunoResearchLaboratories) diluted 1:100 in PBS. Nuclei were stained with propidiumiodide diluted in PBS (0.2-1 μg/ml). Alter three PBS rinses cover slipswere mounted with 50% glycerol. Images were collected with BioRadMRC-600/Nikon Optiphot laser scanning confocal microscope.

Localization of eIF4E, VPg protein and PML in human cells by confocalmicroscopy. HeLa cells seeded on cover slips at about 60% confluencywere washed 3 times with PBS and fixed in 2% cold PFA. Cells wereinfected with 10 MOI of baculoviruses expressing either hisVPg, orhis-tagged domain 2 of HCV helicase or empty bacmid. Cells werecollected at 48 h p.i by centrifugation at 3000 rpm, 5 min, 4° C.,followed by two washes with 500 μl of PBS. Cells were fixed in cold 2%PFA, left for 10 min at Room Temperature, washed two times with 500 μlof PBS and resuspended in 300 μl of PBS. Portions of 100 μl of fixedcells were applied onto clean round coverslips and allowed to attachwhile drying in a lamina flow hood. Next day cover slips were put intowells of 24-wells plate and rehydrated min at room temperature with 500μl portions of PBS. After PBS removal, cells were permeabilized for 10min with cold 0.1% Triton X-100 in PBS, rinsed twice with PBS andblocked with 5% serum in PBS for 30 min at RT. After 2 washes with PBScells were incubated for 1 h at room-temperature with primary antibodies(purified polyclonal anti-VPg, anti-eIF4E (MAb) and anti-PML (MAb)antibodies) diluted 1/100 in PBS, followed by washing and 1 h incubationwith secondary antibody (anti-rabbit FITC or anti-mouse Texas Red)(purchased from Jackson ImmunoResearch Laboratories) diluted 1:100 inPBS. Nuclei were stained with propidium iodide diluted in PBS (0.2-1μg/ml). After three PBS rinses cover slips were mounted with 50%glycerol. Images were collected with BioRad MRC-600/Nikon Optiphot laserscanning confocal microscope.

Effect of VPg protein on cell proliferation.

HeLa, B16, GL26 and IMR90 cells were seeded in 96-wells plate at 1×10⁴cells per well and cultivated overnight. Portion of VPg (0.5-2 μg/well)was diluted into serum-free medium to obtain total volume 10 μl andincubated for 20 min with 0.2 μl TransPass P Protein TransfectionReagent (New England BioLabs) at room temperature. Cells were rinsedwith serum-free medium and covered with 100 μl/well of mixturecontaining serum-free medium and transduction mix. After various time ofincubation at 37° C. time the plates were centrifuged at 1000 rpm for 10min and the supernatants were transferred to the new plate. The releaseof the malate dehydrogenase was measured with the LDH CytotoxicityDetection Kit (Clontech), using multilabeled counter Victorl 412(Wallac) at 490 nm. The level of cytotoxicity was calculated accordingto the LDH kit manual.

Example 2 In Vitro Interaction Assay of Human eIF4E with PVY VPg Proteinand Fragments Thereof

Peptides SEQ ID NO: 2 (VPg), SEQ ID NO: 14 (VPg2), SEQ ID NO: 16 (VPg4),SEQ ID NO: 20 (VPg1), SEQ ID NO: 22 (VPg3) and SEQ ID NO: 18 (VPg5) wereobtained by expression in E. coli as GST-fusion proteins. GST-fusionproteins were produced by standard methods from cDNA sequences (encodingVPg, VPg1, VPg2, VPg3, VPg4 or VPg5) cloned into vector pGEX-4T-1(purchased from Amersham Biosciences).

ELISA technique was used for measuring the peptide's interaction witheIF4E immobilised in wells of 96-well dish. The interaction wasmonitored with an anti-GST antibody.

Human eIF4E, 100 ng in the coating buffer (0.1 M carbonate buffer, pH9.6) was applied to 96-wells micro titer plate for overnight at 4° C.Excess eIF4E was removed and the wells were blocked with 5% milk in thePBST buffer for 1 h at 37° C. The plate was rinsed three times with PBSTand incubated with increasing quantity of the various GST-fusionspeptides in PBST, for 1 h at RT. After two PBST washes the incubationwith anti-GST HRP-conjugated antibody (1:5000) (purchased from AmershamBiosciences) was carried out for 30 min at 37° C. After three PBSTwashes the reaction was developed with peroxides substrate and theabsorbance was measured at 490 nm. Non-specific interactions of GSTprotein and anti-GST antibody with the GST-fusion peptides weresubstracted.

Results are shown in FIG. 1. Among the peptides tested, it appeared thatSEQ ID NO: 22 (VPg3) is the weaker interactor. SEQ ID NO: 20 (VPg1) andSEQ ID NO: 14 (VPg2) showed approximately similar level of interaction,which is not far from VPg itself. The best eIF4E-interacting peptideseems to be SEQ ID NO: 16 (VPg4) and SEQ ID NO: 18 (VPg5), which, athigher concentration functions better than PVY VPg protein itself (SEQID NO: 2).

Example 3 In Vitro Cellular Localisation of eIF4E and PVY VPg Protein(SEQ ID NO: 2) in Insect Cells

Insect cells have been infected with baculovirus containing VPg gene ofPVY (SEQ ID NO: 1). Confocal microscopy analysis revealed that theinitiation factor eIF4E which localized at the beginning of infection inboth compartiments, the nucleus and the cytoplasm (FIG. 2, 12 hpi), at60 hpi timepoint was observed at the cell periphery (FIG. 2, leftcolumns). VPg was observed only in the cytoplasm throughthout theinfection (FIG. 2, VPg column). Merged images showed significantfraction of the elF4E in the nucleus at the beginning of infection butwhich diminished in the course of infection; at 24 hpi, the elF4E wasvisible in the cytoplasm and just at the nucleus periphery and at 60 hpiwas observed in the cytoplasm only, showing significant overlap with theVPg.

To find out if the observed fluctuations in the elF4E localization arespecifically due to the presence of VPg, it was investigated thelocalization of elF4E upon insect cells infection by baculovirus null(bacmid) which is a virus of origin, before insertion of the foreigngene, and also upon infection with baculovirus expressing non-relevantprotein, the domain II of HCV helicase or VPg (FIG. 3). In non-infectedinsect cells elF4E was observed in both cellular compartments (the mostupper panel). At 12 hpi cells infected with all three kinds of virusbehaved similarly, whereas al 60 hpi only cells expressing VPg showedelF4E retention in the cytoplasm (FIG. 3, lowest panel). To obtain morequantitative data, the cell nucleus was separated from the cytoplasm,and elF4E quantity in both compartiments was assessed by Western blot(FIG. 4) followed by densitometry (Table 2 below).

TABLE 2 eIF4E distribution in insect cell cytoplasm and nucleus eIF4E ineIF4E in Hours p.i. baculovirus cytoplasm (%) nucleus (%) HF cells none48.49 51.51 12 h bacmid 46.5 53.5 HCV helicase domain 51.54 48.46 VPg55.5 44.5 hisVPg 53.45 46.55 24 h bacmid 57.83 42.17 HCV helicase domain63.05 36.95 VPg 84.74 15.26 hisVPg 81.3 18.7 36 h bacmid 59.2 40.8 HCVhelicase domain 62.5 37.5 VPg 87.1 12.9 hisVPg 83.33 16.67 48 h bacmid55.99 44.01 HCV helicase domain 57.9 42.1 VPg 73.9 26.1 hisVPg 74.0725.93

Cells infected with the baculovirus expressing his-tagged form of VPgwere included in the experiment. In both non infected as well as cellsinfected for 12 h, comparable level of elF4E were present in thecytoplasm and the nucleus. The nuclear pool of the initiation factordiminished somewhat in the course of infection with bacmid and withhelicase-expressing virus. However, upon expression of both VPg formsthe nuclear pool of the elF4E was severely depleted; at 36 hpi thenuclear pool of elF4E accounted for 13% only of total cellular factor(down from 51-53%).

These experiments demonstrated that (1) VPg expressed alone in thebaculovirus system remains in the cytoplasm; (2) in the presence of VPg,eIF4E is retained in the cytoplasm, resulting in the depletion of theinitiation factor nuclear pool, but no inhibition of cell proliferationor cell death was observed probably because the insects cells containsseveral eIF4E isoforms.

Example 4 In Vitro Cellular Localization of eIF4E and PVY VPg Protein inHela Cells

In order to know if VPg protein of plant virus is able to interact withhuman eIF4E, and resulting in the depletion of the nuclear pool of theinitiation factor, several tranfection experiments were undertaken undervarious conditions. However, no cells expressing VPg could be recovered,suggesting toxic effect of VPg on human cells. Therefore the PVY VPg(SEQ ID NO: 2) was directly translocated into HeLa cell with Pro-Ject™(purchased from Pierce), a cationic lipid mixture which allowsintracellular delivery of proteins (see FIG. 5B). VPg was localized inthe cytoplasm and affected strongly localization of native eIF4E. Theinitiation factor in control HeLa cells was observed in cytoplasm andnucleus. In the presence of VPg the level of eIF4E diminishedsignificantly in the nucleus and overlapped to large extent with thecytoplasm-localized VPg.

Example 5 Effect of PVY VPg Protein on Cell Proliferation In Vitro

The LDH assay, which permits evaluation of cell damage was applied afterVPg delivery to transformed cells, HeLa and B16 and to primarynon-transformed human cells IMR90. Already 1 hour after VPgintracellular delivery both HeLa and B16 showed significant cell damage,Importantly, VPg introduced into primary IMR90 (human fetal lungfibroblasts) did not affect their growth (FIG. 6, IMR90).

The interaction of VPg with the eukaryotic initiation factor eIF4Eresults in the immobilization of the initation factor in the cytoplasmwhich leads to inhibition of cell growth followed by cell death. It isconceivable that the decrease of the eIF4E nuclear pool caused byinteraction with VPg is accompanied by inhibition of the cytoplasmictransport of the messenger RNA of the pro-proliferative proteins,leading to the inhibition of cell growth with ensuing cell death.

Example 6 Efficacy of PVY VPg Protein on Cell Proliferation In Vivo

To study anticancer effect of PVY VPg protein (SEQ ID NO: 2), in vivoexperiences have been undertaken. C57BL/6 mice were inoculated with GL26cells inducing the mouse glioma tumours or with B16-ova melanoma cells.

GL26 tumours were established by injecting portions of 0.5×10⁶ GL26cells in 100 μl foetal bovine serum into the right quadriceps of theadult female C57BL/6 mice. About two weeks later, during the exponentialphase of glioma tumour growth, the VPg (25 μg in 0.9% NaCl) or 0.9% NaCl(50 μl) (buffer) were administered by electroporation to 2 groups of 5mice, leaving the control group of mice untreated. VPg and buffertreated mice were anaesthetized by intraperitoneal injection of 250 μlof a solution containing 400 μl of Imalgen 1000, 2% Rompun (both fromCentravet, Lapalisse, France) and 0.9% NaCl. Electroporation conditionswere the following: 5×100 μsec pulses of the 800 volts from electrosquare porator T820 (BTX, San Diego, Calif., U.S.A), at 1 Hz frequency.The delay between pulses was one second. The animals were kept warm (37°C.) until they recovered from anaesthesia. The volume of the tumours wasmeasured in mm³ every 2 or 3 days by slide calliper and compared withnormally growing glioma tumors of the control untreated group of mice.

B16-ova melanoma tumours were established by injection of 5×10⁵ cellssuspended in PBS into the left thighs of the C57BL/6 mice (two groups of8 animals). After 12 days, when palpable tumors were formed, the tumoursof the control group were injected with Transpass P mixed with thebuffer. Second group had the injections of 50 micrograms of VPgtransduced with Transpass P, every day for 6 days. The kinetics oftumour growth was followed by measuring every day the tumor diameter.

Results are shown in FIGS. 7 and 8. Administration of VPg slowed down orinhibited the growth of the tumours in comparison with control groups.This shows that VPg has indeed an inhibitory effect on tumourproliferation.

REFERENCES

-   Boguszewska-Chachulska A M, Krawczyk M, Stankiewicz A, Gozdek A,    Haenni A L, Strokovskaya L. (2004) Direct fluorometric measurement    of hepatitis C virus helicase activity. FEBS Lett. 567:253-6.-   Chaudhry Y, Nayak A, Bordeleau M E, Tanaka J, Pelletier J, Belsham G    J, Roberts L O, Goodfellow I G. (2006) Caliciviruses differ in their    functional requirements for eIF4F components. J. Biol. Chem.    281:25315-25.-   Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G,    Prochiantz A. (1996) Cell internalization of the third helix of the    Antennapedia homeodomain is receptor-independent. J. Biol. Chem.    271:18188-93.-   Duprat A, Caranta C, Revers F, Menand B, Browning K S,    Robaglia C. (2002) The Arabidopsis eukaryotic initiation factor    (iso)4E is dispensable for plant growth but required for    susceptibility to potyviruses. Plant J. 32:927-34.-   Elliott G and O'Hare P. (1997) Intercellular trafficking and protein    delivery by a herpesvirus structural protein. Cell 88:223-33.-   Fellers, J, Wan J, Hong Y, Collins G B, and Hunt A G. (1998). In    vitro interactions between a potyvirus-encoded, genome-linked    protein and RNA-dependent RNA polymerase. J. Gen. Virol.    79:2043-2049.-   Goodfellow I, Chaudhry Y, Gioldasi I, Gerondopoulos A, Natoni A,    Labrie L, Laliberte J F, Roberts L. (2005) Calicivirus translation    initiation requires an interaction between VPg and eIF4E. EMBO Rep.    6: 968-972.-   Grzela R, Strokovska L, Andrieu J P, Dublet B, Zagorski W,    Chroboczek J. (2006) Potyvirus terminal protein VPg, effector of    host eukaryotic initiation factor eIF4E Biochimie 88:887-96.-   Herbert T P, Brierley I, Brown T D. (1997) Identification of a    protein linked to the genomic and subgenomic mRNAs of feline    calicivirus and its role in translation. J. Gen Virol. 78:1033-40.-   Jans D A. (1994) Nuclear signaling pathways for peptide ligands and    their membrane receptors? Faseb J. 8:841-847-   Kang B C, Yearn I, Frantz J D, Murphy J F, Jahn M M. (2005) The pvr1    locus in Capsicum encodes a translation initiation factor eIF4E that    interacts with Tobacco etch virus VPg. Plant J. 42:392-405.-   Kokryakov V N, Harwig S S, Panyutich E A, Shevchenko A A, Aleshina G    M, Shamova O V, Korneva H A, Lehrer R1. (1993) Protegrins: leukocyte    antimicrobial peptides that combine features of corticostatic    defensins and tachyplesins. FEBS Lett. 327:231-6.-   Lai H K, Borden K L, (2000) The promyelocytic leukemia (PML) protein    suppresses cyclin D1 protein production by altering the nuclear    cytoplasmic distribution of cyclin D1 mRNA. Oncogene 19:1623-34.-   Lejbkowicz F, Goyer C, Darveau A, Neron S, Lemieux R,    Sonenberg N. (1992) A fraction of the mRNA 5′ cap-binding protein,    eukaryotic initiation factor 4E, localizes to the nucleus. Proc Natl    Acad Sci USA. 89:9612-6.-   Lellis A D, Kasschau K D, Whitham S A, Carrington J C. (2002)    Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an    essential role for eIF(iso)₄E during potyvirus infection. Curr Biol.    12:1046-51-   Leonard S, Plante D, Wittmann S, Daigneault N, Fortin M G, Laliberte    J F. (2000) Complex formation between potyvirus VPg and translation    eukaryotic initiation factor 4E correlates with virus    infectivity. J. Virol. 74:7730-7.-   Murphy J F, Rychlik W, Rhoads R E, Hunt A G, Shaw J G. (1991) A    tyrosine residue in the small nuclear inclusion protein of tobacco    vein mottling virus links the VPg to the viral RNA. J. Virol.    65:511-3.-   O'Reilly D R, Miller L K and Luckow V A. (1992) Baculovirus    Expression Vectors: A Laboratory Manual. (New York, N.Y.: W.H.    Freemean and Company).-   Riechmann J L, Lain S, Garcia J A. (1989) The genome-linked protein    and 5′ end RNA sequence of plum pox potyvirus. J Gen Virol.    70:2785-9.-   Rosenwald I B, Kaspar R, Rousseau D, Gehrke L, Leboulch P, Chen J J,    Schmidt E V, Sonenberg N, London I M. (1995) Eukaryotic translation    initiation factor 4E regulates expression of cyclin D1 at    transcriptional and post-transcriptional levels. J Biol Chem.    270:21176-80.-   Rousseau D, Kaspar R, Rosenwald I, Gehrke L, Sonenberg N. (1996)    Translation initiation of ornithine decarboxylase and    nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells    overexpressing eukaryotic initiation factor 4E. Proc Natl Acad Sci    USA. 93:1065-70.-   Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine    W A, Rosen C A. (1989) Structural and functional characterization of    human immunodeficiency virus tat protein. J. Virol. 63:1-8.-   Ruggero D and Pandolfi P P. (2003) Does the ribosome translate    cancer? Nat. Rev. Cancer. 3:179-92.-   Schaad M C, Lellis A D, and Carrington J C. (1997) VPg of tobacco    etch potyvirus is a host genotype-specific determinant for    long-distance movement. J. Virol. 71:8624-8631.-   Shantz L M, Pegg A E. (1994) Overproduction of ornithine    decarboxylase caused by relief of translational repression s    associated with neoplastic transformation. Cancer Res. 54:2313-6.-   Sonenberg N, Gingras A C. (1998) The mRNA 5′ cap-binding protein    eIF4E and control of cell growth. Curr Opin Cell Biol. 10:268-75.-   Wittmann S, Chatel H, Fortin M G, Laliberte J F. (1997) Interaction    of the viral protein genome linked of turnip mosaic potyvirus with    the translational eukaryotic initiation factor (iso) 4E of    Arabidopsis thaliana using the yeast two-hybrid system. Virology.    234:84-92.

1. An isolated and purified protein comprising or consisting of a VPgprotein or a fragment thereof of at least 8 amino acids or an analogthereof: (i) having the ability to bind an eukaryotic initiation factoreIF4E, preferably a mammal initiation factor eIF4E and more preferably ahuman initiation factor eIF4E, (ii) said VPg protein, fragment thereofor analog thereof not comprising the amino acid motif YxxxxLØ, wherein xis any amino acid, Y is tyrosine (Tyr), L is leucine (Leu) and Ø isleucine (Leu), methionine (Met) or phenylalanine (Phe), as a medicament.2. The protein according to claim 1, characterized in that it consistsof the VPg protein of SEQ ID NO:
 2. 3. The protein according to claim 1,characterized in that it consists of a VPg protein analog exhibiting atleast 20% sequence identity, or at least 45%, sequence similarity withthe full-length of SEQ ID NO:
 2. 4. The protein according to claim 3,characterized in that said protein analog is selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29 or SEQ ID NO:30.
 5. A fragment of a protein according to claim 1, characterized inthat it comprises or consists of at least 8 contiguous amino acids ofsaid VPg proteins.
 6. The fragment of a protein according to claim 5,characterized in that said fragment is selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20or SEQ ID NO:
 22. 7. The fragment of a protein according to claim 6,characterized in it consists of an analog of the fragment defined by SEQID NO: 16, exhibiting at least 60% sequence identity, or at least 80%,sequence similarity with the full-length of SEQ ID NO:
 16. 8. Thefragment of a protein according to claim 6, characterized in that itconsists of an analog of the fragment defined by SEQ ID NO: 18,exhibiting at least 60% sequence identity, or at least 80%, sequencesimilarity with the full-length of SEQ ID NO:
 18. 9. The fragment of aprotein according to claim 5, characterized in that it comprises orconsists of at least 8, 10, 15, 20, 25 or 26 contiguous amino acids ofSEQ ID NO:
 16. 10. The protein, fragment thereof or analog thereofaccording to claim 1, as an anti-cancer agent.
 11. The protein, fragmentthereof or analog thereof according to claim 1 for use in treatingcancer, preferably glioma, melanoma or a colon or lung cancer, in whichthe cancer cells express the initiation factor eIF4E.
 12. Use of aprotein, fragment thereof or analog thereof according to claim 1 for themanufacture of a medicament for treating cancer, in which the initiationfactor eIF4E is expressed.
 13. Method of screening for compounds whichmimic the binding specificity of the a VPg protein of SEQ ID NO: 2 withan isolated and purified initiation factor eIF4E of a subject,characterized in that it comprises the steps of i) contacting said eIF4Ewith a candidate compound and a protein, fragment thereof or analogthereof according to claim 1, and ii) detecting if the candidatecompound inhibits the interaction between eIF4E and said protein,fragment thereof or analog thereof.
 14. Use of a recombinant vectorexpressing a nucleic acid encoding a protein, fragment thereof or analogthereof according to claim 1, for the preparation of a medicament forpreventing or treating cancer.
 15. Use according to claim 14,characterized in that the nucleic acid is selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or
 21. 16. Apharmaceutical composition comprising a protein, fragment thereof oranalog thereof according to claim 1, and a pharmaceutically acceptablecarrier.
 17. The composition according to claim 16, characterized inthat the pharmaceutically acceptable carrier is a cationic lipid. 18.The composition according to claim 16 for use in treating cancer, inwhich the initiation factor eIF4E is expressed.
 19. Host celltransfected to express a protein, fragment thereof or analog thereofaccording to claim
 1. 20. Use of an isolated and purified initiationfactor eIF4E to screen compounds that mimic the binding specificity ofthe VPg protein of SEQ ID NO: 2 with said eIF4E.